Phase I studies of peptide vaccine cocktails derived from GPC3, WDRPUH and NEIL3 for advanced hepatocellular carcinoma.

Aim: Two peptide cocktail vaccines using glypican-3, WD-repeat-containing protein up-regulated in hepatocellular carcinoma (HCC) and nei endonuclease VIII-like three epitopes were evaluated in advanced HCC in two Phase I studies. Patients & methods: Study 1 evaluated dose-limiting toxicities (DLTs) of peptides 1-3 (HLA-A24-restricted) and study 2 evaluated DLTs of peptides 1-6 (HLA-A24 or A02-restricted). Results: Overall, 18 and 14 patients were enrolled in studies 1 and 2, respectively. No DLTs were observed up to 7.1 mg of the vaccine cocktail. No complete response/partial response was observed. Stable disease was reported in nine and five patients with a disease control rate of 52.9% and 35.7% in studies 1 and 2, respectively. Conclusion: Both vaccines showed good tolerability and potential usefulness against HCC. Clinical trial registration: JapicCTI-121933; JapicCTI-142477.

(p)ppGpp-Dependent Regulation of the Nucleotide Hydrolase PpnN Confers Complement Resistance in Salmonella enterica Serovar Typhimurium.

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria, including Salmonella enterica serovar Typhimurium. S Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues, resulting in severe clinical outcomes. During infection, S Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5'-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane, known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

Production of a polyclonal antibody against inosine-uridine preferring nucleoside hydrolase of Acanthamoeba castellanii and its access to diagnosis of Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a rare disease but its prevalence throughout the globe continues to grow, primarily due to increased contact lens usage. Since early-stage symptoms associated with AK closely resemble those from other corneal infections, accurate diagnosis is difficult and this often results in delayed treatment and exacerbation of the disease, which can lead to permanent visual impairment. Accordingly, developing a rapid Acanthamoeba-specific diagnostic method is highly desired. In the present study, a rapid and differential method for AK diagnosis was developed using the secretory proteins derived from the pathogenic Acanthamoeba. Among the vast quantities of proteins secreted by the pathogenic Acanthamoeba, an open reading frame of the inosine-uridine preferring nucleoside hydrolase (IPNH) gene was obtained. After expressing and purifying the IPNH protein using the pGEX 4T-3 vector system, mice were immunized with the purified proteins for polyclonal antibody generation. Western blot was performed using protein lysates of the human corneal cell, non-pathogenic amoeba, pathogenic amoeba, and clinical amoeba isolate along with lysates from other causes of keratitis such as Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium solani to confirm Acanthamoeba-specificity. Western blot using the polyclonal IPNH antibody revealed that IPNH was Acanthamoeba-specific since these proteins were only observed in lysates of Acanthamoeba origin or its culture media. Our findings indicate that the IPNH antibody of Acanthamoeba may serve as a potential agent for rapid and differential AK diagnosis.

Nucleoside Hydrolase NH 36: A Vital Enzyme for the Leishmania Genus in the Development of T-Cell Epitope Cross-Protective Vaccines.

NH36 is a vital enzyme of the DNA metabolism and a specific target for anti-Leishmania chemotherapy. We developed second-generation vaccines composed of the FML complex or its main native antigen, the NH36 nucleoside hydrolase of Leishmania (L.) donovani and saponin, and a DNA vaccine containing the NH36 gene. All these vaccines were effective in prophylaxis and treatment of mice and dog visceral leishmaniasis (VL). The FML-saponin vaccine became the first licensed veterinary vaccine against leishmaniasis (Leishmune®) which reduced the incidence of human and canine VL in endemic areas. The NH36, DNA or recombinant protein vaccines induced a Th1 CD4+IFN-γ+ mediated protection in mice. Efficacy against VL was mediated by a CD4+TNF-α T lymphocyte response against the NH36-F3 domain, while against tegumentary leishmaniasis (TL) a CD8+ T lymphocyte response to F1 was also required. These domains were 36-41 % more protective than NH36, and a recombinant F1F3 chimera was 21% stronger than the domains, promoting a 99.8% reduction of the parasite load. We also identified the most immunogenic NH36 domains and epitopes for PBMC of active human VL, cured or asymptomatic and DTH+ patients. Currently, the NH36 subunit recombinant vaccine is turning into a multi-epitope T cell synthetic vaccine against VL and TL.

NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.

Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.

Prediction and analysis of promiscuous T cell-epitopes derived from the vaccine candidate antigens of Leishmania donovani binding to MHC class-II alleles using in silico approach.

Visceral leishmaniasis is a dreadful infectious disease and caused by the intracellular protozoan parasites, Leishmania donovani and Leishmania infantum. Despite extensive efforts for developing effective prophylactic vaccine, still no vaccine is available against leishmaniasis. However, advancement in immunoinformatics methods generated new dimension in peptide based vaccine development. The present study was aimed to identify T-cell epitopes from the vaccine candidate antigens like Lipophosphogylcan-3(LPG-3) and Nucleoside hydrolase (NH) from the L. donovani using in silico methods. Available best tools were used for the identification of promiscuous peptides for MHC class-II alleles. A total of 34 promiscuous peptides from LPG-3, 3 from NH were identified on the basis of their 100% binding affinity towards all six HLA alleles, taken in this study. These peptides were further checked computationally to know their IFN-γ and IL4 inducing potential and nine peptides were identified. Peptide binding interactions with predominant HLA alleles were done by docking. Out of nine docked promiscuous peptides, only two peptides (QESRILRVIKKKLVR, RILRVIKKKLVRKTL), from LPG-3 and one peptide (FDKFWCLVIDALKRI) from NH showed lowest binding energy with all six alleles. These promiscuous T-cell epitopes were predicted on the basis of their antigenicity, hydrophobicity, potential immune response and docking scores. The immunogenicity of predicted promiscuous peptides might be used for subunit vaccine development with immune-modulating adjuvants.

Expression, purification, immunogenicity and protective efficacy of a recombinant nucleoside hydrolase from Leishmania donovani, a vaccine candidate for preventing cutaneous leishmaniasis.

The nucleoside hydrolase gene from Leishmania donovani was cloned and expressed in Escherichia coli as a full length 36-kDa protein (LdNH36). Following lysis and extraction, the protein was purified by anion exchange and gel filtration chromatography. The purified protein had a molecular mass of approximately 36-kDa and was confirmed to be >99% pure. Using a nucleoside hydrolase assay, the protein was found to exhibit a Km of 741 ± 246 µM. Protein integrity was confirmed by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE), mass spectrometry (MS), and enzymatic assay. Analysis of antibody levels from immunized mice indicated that LdNH36 alone or in a stable emulsion with the Toll-like receptor-4 ligand glucopyranosyl lipid adjuvant (GLA-SE) as immunostimulant induced high levels of antigen-specific IgG antibodies. The cellular immune response indicated a Th1 response in mice immunized with LdNH36, but only when formulated with GLA-SE. Mice immunized with the LdNH36 antigen in combination with the GLA-SE adjuvant and challenged with Leishmania mexicana showed significant reductions (>20 fold) in parasite burden, confirming the protective efficacy of this vaccine candidate.

Deficiency of base excision repair enzyme NEIL3 drives increased predisposition to autoimmunity.

Alterations in the apoptosis of immune cells have been associated with autoimmunity. Here, we have identified a homozygous missense mutation in the gene encoding the base excision repair enzyme Nei endonuclease VIII-like 3 (NEIL3) that abolished enzymatic activity in 3 siblings from a consanguineous family. The NEIL3 mutation was associated with fatal recurrent infections, severe autoimmunity, hypogammaglobulinemia, and impaired B cell function in these individuals. The same homozygous NEIL3 mutation was also identified in an asymptomatic individual who exhibited elevated levels of serum autoantibodies and defective peripheral B cell tolerance, but normal B cell function. Further analysis of the patients revealed an absence of LPS-responsive beige-like anchor (LRBA) protein expression, a known cause of immunodeficiency. We next examined the contribution of NEIL3 to the maintenance of self-tolerance in Neil3-/- mice. Although Neil3-/- mice displayed normal B cell function, they exhibited elevated serum levels of autoantibodies and developed nephritis following treatment with poly(I:C) to mimic microbial stimulation. In Neil3-/- mice, splenic T and B cells as well as germinal center B cells from Peyer's patches showed marked increases in apoptosis and cell death, indicating the potential release of self-antigens that favor autoimmunity. These findings demonstrate that deficiency in NEIL3 is associated with increased lymphocyte apoptosis, autoantibodies, and predisposition to autoimmunity.

Genetic cause of immune dysregulation - one gene or two?

Some autoimmune disorders are monogenetic diseases; however, clinical manifestations among individuals vary, despite the presence of identical mutations in the disease-causing gene. In this issue of the JCI, Massaad and colleagues characterized a seemingly monogenic autoimmune disorder in a family that was linked to homozygous loss-of-function mutations in the gene encoding the endonuclease Nei endonuclease VIII-like 3 (NEIL3), which has not been previously associated with autoimmunity. The identification of an unrelated healthy individual with the same homozygous mutation spurred more in-depth analysis of the data and revealed the presence of a second mutation in a known autoimmune-associated gene. Animals lacking Neil3 had no overt phenotype, but were predisposed to autoantibody production and nephritis following exposure to the TLR3 ligand poly(I:C). Together, these results support further evaluation of the drivers of autoimmunity in supposedly monogenic disorders.

SOBIR1 contributes to non-host resistance to Magnaporthe oryzae in Arabidopsis.

The rate of entry of Magnaporthe oryzae into Arabidopsis pen2 sobir1 plants was significantly higher than that into pen2 plants. The length of the infection hyphae in pen2 sobir1 plants was significantly longer than that in pen2 plants. These results suggest that SOBIR1 is involved in both penetration and post-penetration resistance to M. oryzae in Arabidopsis.

Expression and purification of an engineered, yeast-expressed Leishmania donovani nucleoside hydrolase with immunogenic properties.

Leishmania donovani is the major cause of visceral leishmaniasis (kala-azar), now recognized as the parasitic disease with the highest level of mortality second only to malaria. No human vaccine is currently available. A 36 kDa L. donovani nucleoside hydrolase (LdNH36) surface protein has been previously identified as a potential vaccine candidate antigen. Here we present data on the expression of LdNH36 in Pichia pastoris and its purification at the 20 L scale to establish suitability for future pilot scale manufacturing. To improve efficiency of process development and ensure reproducibility, 4 N-linked glycosylation sites shown to contribute to heterogeneous high-mannose glycosylation were mutated to glutamine residues. The mutant LdNH36 (LdNH36-dg2) was expressed and purified to homogeneity. Size exclusion chromatography and light scattering demonstrated that LdNH36-dg2 existed as a tetramer in solution, similar to the wild-type recombinant L. major nucleoside hydrolase. The amino acid mutations do not affect the tetrameric interface as confirmed by theoretical modeling, and the mutated amino acids are located outside the major immunogenic domain. Immunogenic properties of the LdNH36-dg2 recombinant protein were evaluated in BALB/c mice using formulations that included a synthetic CpG oligodeoxynucleotide, together with a microparticle delivery platform (poly(lactic-co-glycolic acid)). Mice exhibited high levels of IgG1, IgG2a, and IgG2b antibodies that were reactive to both LdNH36-dg2 and LdNH36 wild-type. While the point mutations did affect the hydrolase activity of the enzyme, the IgG antibodies elicited by LdNH36-dg2 were shown to inhibit the hydrolase activity of the wild-type LdNH36. The results indicate that LdNH36-dg2 as expressed in and purified from P. pastoris is suitable for further scale-up, manufacturing, and testing in support of future first-in-humans phase 1 clinical trials.

Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 µm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.

Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood.

Streptococcus agalactiae (Group B Streptococcus) is a commensal of the human intestine and vagina of adult women but is the leading cause of invasive infection in neonates. This Gram-positive bacterium displays a set of virulence-associated surface proteins involved in the interaction with the host, such as adhesion to host cells, invasion of tissues, or subversion of the immune system. In this study, we characterized a cell wall-localized protein as an ecto-5'-nucleoside diphosphate phosphohydrolase (NudP) involved in the degradation of extracellular nucleotides which are central mediators of the immune response. Biochemical characterization of recombinant NudP revealed a Mn(2+)-dependent ecto-5'-nucleotidase activity on ribo- and deoxyribonucleoside 5'-mono- and 5'-diphosphates with a substrate specificity different from that of known orthologous enzymes. Deletion of the gene coding the housekeeping enzyme sortase A led to the release of NudP into the culture supernatant, confirming that this enzyme is anchored to the cell wall by its non-canonical LPXTN motif. The NudP ecto-5'-nucleotidase activity is reminiscent of the reactions performed by the mammalian ectonucleotidases CD39 and CD73 involved in regulating the extracellular level of ATP and adenosine. We further demonstrated that the absence of NudP activity decreases bacterial survival in mouse blood, a process dependent on extracellular adenosine. In vivo assays in animal models of infection showed that NudP activity is critical for virulence. These results demonstrate that Group B Streptococcus expresses a specific ecto-5'-nucleotidase necessary for its pathogenicity and highlight the diversity of reactions performed by this enzyme family. These results suggest that bacterial pathogens have developed specialized strategies to subvert the mammalian immune response controlled by the extracellular nucleotide signaling pathways.

Glutathione and tryptophan metabolism are required for Arabidopsis immunity during the hypersensitive response to hemibiotrophs.

The hypersensitive response (HR) is a type of strong immune response found in plants that is accompanied by localized cell death. However, it is unclear how HR can block a broad range of pathogens with different infective modes. In this study, we report that γ-glutamylcysteine synthetase GSH1, which is critical for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to HR and block development of fungal pathogens with hemibiotrophic infective modes. We found that GSH1 is involved in the penetration2 (PEN2)-based entry control of the nonadapted hemibiotroph Colletotrichum gloeosporioides. However, Arabidopsis mutants specifically defective in entry control terminated further growth of the pathogen in the presence of HR cell death, whereas gsh1 mutants supported pathogen invasive growth in planta, demonstrating the requirement of GSH1 for postinvasive nonhost resistance. Remarkably, on the basis of the phenotypic and metabolic analysis of Arabidopsis mutants defective in Trp metabolism, we showed that biosynthesis of Trp-derived phytochemicals is also essential for resistance to C. gloeosporioides during postinvasive HR. By contrast, GSH1 and these metabolites are likely to be dispensable for the induction of cell death during postinvasive HR. Furthermore, the resistance to Ralstonia solanacearum 1/resistance to Pseudomonas syringae 4 dual Resistance gene-dependent immunity of Arabidopsis to the adapted hemibiotroph shared GSH1 and cytochromes P450 CYP79B2/CYP79B3 with postinvasive nonhost resistance, whereas resistance to P. syringae pv. maculicola 1 and resistance to P. syringae 2-based Resistance gene resistance against bacterial pathogens did not. These data suggest that the synthesis of glutathione and Trp-derived metabolites during HR play crucial roles in terminating the invasive growth of both nonadapted and adapted hemibiotrophs.

A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies.

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30 kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.

Adaptive immunity against Leishmania nucleoside hydrolase maps its c-terminal domain as the target of the CD4+ T cell-driven protective response.

Nucleoside hydrolases (NHs) show homology among parasite protozoa, fungi and bacteria. They are vital protagonists in the establishment of early infection and, therefore, are excellent candidates for the pathogen recognition by adaptive immune responses. Immune protection against NHs would prevent disease at the early infection of several pathogens. We have identified the domain of the NH of L. donovani (NH36) responsible for its immunogenicity and protective efficacy against murine visceral leishmaniasis (VL). Using recombinant generated peptides covering the whole NH36 sequence and saponin we demonstrate that protection against L. chagasi is related to its C-terminal domain (amino-acids 199-314) and is mediated mainly by a CD4+ T cell driven response with a lower contribution of CD8+ T cells. Immunization with this peptide exceeds in 36.73±12.33% the protective response induced by the cognate NH36 protein. Increases in IgM, IgG2a, IgG1 and IgG2b antibodies, CD4+ T cell proportions, IFN-γ secretion, ratios of IFN-γ/IL-10 producing CD4+ and CD8+ T cells and percents of antibody binding inhibition by synthetic predicted epitopes were detected in F3 vaccinated mice. The increases in DTH and in ratios of TNFα/IL-10 CD4+ producing cells were however the strong correlates of protection which was confirmed by in vivo depletion with monoclonal antibodies, algorithm predicted CD4 and CD8 epitopes and a pronounced decrease in parasite load (90.5-88.23%; p = 0.011) that was long-lasting. No decrease in parasite load was detected after vaccination with the N-domain of NH36, in spite of the induction of IFN-γ/IL-10 expression by CD4+ T cells after challenge. Both peptides reduced the size of footpad lesions, but only the C-domain reduced the parasite load of mice challenged with L. amazonensis. The identification of the target of the immune response to NH36 represents a basis for the rationale development of a bivalent vaccine against leishmaniasis and for multivalent vaccines against NHs-dependent pathogens.

A combination DNA vaccine encoding nucleoside hydrolase 36 and glycoproteine 63 protects female but not male hamsters against Leishmania mexicana.

Leishmaniasis is a group of diseases caused by protozoan parasites of the Leishmania genus. Previous studies have shown that a DNA vaccine encoding Leishmania donovani antigen nucleoside hydrolase 36 and L. mexicana glycoprotein 63 is protective in mice. We investigated here the efficacy of this DNA vaccine to induce protection in golden hamsters. Male hamsters were more susceptible to infection by Leishmania mexicana than females. Following immunization with two doses of the DNA vaccine, only females resulted protected while males developed normal lesions.

Evaluation of immune responses and protection induced by A2 and nucleoside hydrolase (NH) DNA vaccines against Leishmania chagasi and Leishmania amazonensis experimental infections.

Several antigens have been tested as vaccine candidates against Leishmania infections but controversial results have been reported when different antigens are co-administered in combined vaccination protocols. Immunization with A2 or nucleoside hydrolase (NH) antigens was previously shown to induce Th1 immune responses and protection in BALB/c mice against Leishmania donovani and L. amazonensis (A2) or L. donovani and L. mexicana (NH) infections. In this work, we investigated the protective efficacy of A2 and NH DNA vaccines, in BALB/c mice, against L. amazonensis or L. chagasi challenge infection. Immunization with either A2 (A2-pCDNA3) or NH (NH-VR1012) DNA induced an elevated IFN-gamma production before infection; however, only A2 DNA immunized mice were protected against both Leishmania species and displayed a sustained IFN-gamma production and very low IL-4 and IL-10 levels, after challenge. Mice immunized with NH/A2 DNA produced higher levels of IFN-gamma in response to both specific recombinant proteins (rNH or rA2), but displayed higher IL-4 and IL-10 levels and increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals. These data extend the characterization of the immune responses induced by NH and A2 antigens as potential candidates to compose a defined vaccine and indicate that a highly polarized type 1 immune response is required for improvement of protective levels of combined vaccines against both L. amazonensis and L. chagasi infections.

Protection of susceptible BALB/c mice from challenge with Leishmania major by nucleoside hydrolase, a soluble exo-antigen of Leishmania.

Leishmania major culture-derived, soluble, exogenous antigens have been shown to be a source of vaccine targets for the parasite. We have previously reported that L. major culture-derived, soluble, exogenous antigens can immunize BALB/c mice against challenge with L. major. However, the molecule(s) involved in this protection was not known. We describe the potential of one component of soluble exogenous antigens (recombinant nucleoside hydrolase) to vaccinate mice against challenge with L. major. We found that recombinant nucleoside hydrolase vaccinated BALB/c mice against a subsequent challenge with L. major. Protection was manifested by a significant decrease in lesion size (as much as a 30-fold reduction) and parasite burden (as much as a 71-fold reduction). Protection was achieved whether recombinant nucleoside hydrolase was administered to mice in the presence or absence of adjuvant (interleukin-12). Finally, protection was accompanied by an increase in interferon-gamma production but a decrease in interleukin-10 production by vaccinated animals in response to challenge with L. major.

Induction of cytokines by toxins that have an identical RNA N-glycosidase activity: Shiga toxin, ricin, and modeccin.

Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.